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We thank V. Denic, M. Pleska, D. Siekhaus, and B. Stern for
in-depth comments on the manuscript; A. W. Murray, P. Cluzel,
B. R. Levin, and J. M. Marko for helpful discussions; and members
of the Guet laboratory for comments. G. T. acknowledges Austrian
Science Fund (FWF) grant P28844. D.J.K. was supported by Swiss
National Science Foundation grant 31003A_149267 to M. Ackermann.
E.B. was supported by Consejo Nacional de Ciencia y Tecnologia
de Mexico (CONACYT) and the Pew Foundation Latin American
Fellows Program. All data and code to understand and assess the
conclusions of this research are available in the main text,
supplementary materials, and at the following repository: http://dx.
doi.org/10.15479/AT:ISTA:53. T.B. and C.C.G. designed the
experiments; T.B. and K. T. performed the experiments; A.M.C.A.
and G. T. developed the model; A.M.C.A., T.B., G. T., K. T., and
C.C.G. analyzed the data; A.M.C.A., E.B., and R.H. wrote analysis
software and scripts; D.J.K. designed and manufactured microfluidic
wafers; and A.M.C.A., T.B., G. T., and C.C.G. wrote the paper.
Materials and Methods
Figs. S1 to S19
Tables S1 to S11
Movies S1 to S3
23 February 2016; resubmitted 30 September 2016
Accepted 13 March 2017
Neonatal acquisition of Clostridia
species protects against colonization
by bacterial pathogens
Yun-Gi Kim,1,2*†‡ Kei Sakamoto,1,2 Sang-Uk Seo,1,2§ Joseph M. Pickard,1,2
Merritt G. Gillilland III,3 Nicholas A. Pudlo,4 Matthew Hoostal,3 Xue Li,3
Thomas D. Wang,5 Taylor Feehley,6 Andrew T. Stefka,6 Thomas M. Schmidt,3,4
Eric C. Martens,4 Shinji Fukuda,7,8 Naohiro Inohara,1
Cathryn R. Nagler,6 Gabriel Núñez1,2†
The high susceptibility of neonates to infections has been assumed to be due to
immaturity of the immune system, but the mechanism remains unclear. By colonizing adult
germ-free mice with the cecal contents of neonatal and adult mice, we show that the
neonatal microbiota is unable to prevent colonization by two bacterial pathogens that
cause mortality in neonates. The lack of colonization resistance occurred when
Clostridiales were absent in the neonatal microbiota. Administration of Clostridiales, but
not Bacteroidales, protected neonatal mice from pathogen infection and abrogated
intestinal pathology upon pathogen challenge. Depletion of Clostridiales also abolished
colonization resistance in adult mice. The neonatal bacteria enhanced the ability of
protective Clostridiales to colonize the gut.
Newborns and children less than 1 year old are highly susceptible to frequent infection by orally acquired bacterial pathogens (1, 2). Susceptibility to intestinal infections in eonates has been generally ascribed to
immaturity of the innate and adaptive immune
systems; however, additional factors may play a
role because immune responses to different stimu-
li are highly variable among neonates (3). The
gut microbiota is important to the development
of the immune system (4, 5). For example, gut
microbiota–induced local responses, such as se-
cretory immunoglobulin A, as well as local T
helper 17 cells and regulatory T cells, contrib-
ute to gut homeostasis. Another major attribute
of the microbiota is to protect the host against
colonization by exogenous pathogens, a function
termed “colonization resistance” (4, 6). The gut
microbiota of neonates is less diverse than that
of adult individuals and tends to lack Clostridiales
and Bacteroidales, the dominant taxa found in
the adult intestine (7, 8).
To compare the function of the neonatal and
the adult microbiota in colonization resistance
against pathogens independently of the age of
the host, we colonized age-matched adult germ-
free (GF) mice with the cecal contents of neo-
natal mice or adult (7-week-old) mice and kept
the reconstituted mice in isolators to prevent con-
tamination with exogenous bacteria. Analysis of
the 16S ribosomal RNA (rRNA) gene of the fecal
microbiota 21 days after reconstitution revealed
that the bacterial composition of adult GF mice
colonized with the microbiota from 4-day-old
mice resembled that of the donor and was
dominated by facultative anaerobes including
Lactobacillaceae but devoid of Clostridiales and
Bacteroidales (Fig. 1A and fig. S1A). The micro-
biota of GF mice reconstituted with feces from
12-day-old mice was dominated by operational
taxonomic units (OTUs) belonging to the En-
terobacteriaceae and Lactobacillaceae families,
and few OTUs belonging to the Lachnospiraceae
family compared with that of 16-day-old and
adult mice (Fig. 1A and fig. S1, A and B). In con-
trast, strict anaerobic bacteria with a large number
of Clostridiales OTUs belonging to Lachnospir-
aceae and Ruminococcaceae families, as well as
Porphyromonadaceae and unclassified Bacteroi-
dales, were prevalent in GF mice colonized with
the cecal contents of 16-day-old or adult mice
(Fig. 1A and fig. S1, A and B).
Consistently, there was a greater diversity in
the microbiota of GF mice reconstituted with
16-day-old and adult mice than in mice colonized
with the cecal contents of 4-day-old and 12-day-old
mice (Fig. 1B). To assess the ability of the different
microbiotas to control pathogen replication in the
intestine in the absence of systemic invasion, we
intragastrically infected reconstituted GF mice
with a Salmonella enterica serovar Typhimurium
(S. Typhimurium) mutant deficient in the type
III secretion system (T3SS) encoded by Salmonella
pathogenicity island 2 (DspiA), which replicates
normally in the intestine but is deficient in systemic spread (9, 10). We found that ~50% of GF
mice colonized with the microbiota of 4-day-old
mice succumbed to S. Typhimurium infection,
whereas all GF mice colonized with the adult
microbiota survived (Fig. 1C).
The increased mortality of GF mice harboring a microbiota from 4-day-old mice was associated with marked intestinal cell damage,
submucosal edema, and inflammatory cell infiltrates in the cecum, which were absent in
GF mice colonized with the microbiota of adult
mice (Fig. 1, D and E). Consistent with these
findings, ~80% of 7-day-old mice infected with
S. Typhimurium DspiA succumbed, whereas all
adult mice survived the infection (fig. S2). Notably, GF mice colonized with the microbiota
1Department of Pathology, University of Michigan Medical
School, Ann Arbor, MI 48109, USA. 2Comprehensive Cancer
Center, University of Michigan Medical School, Ann Arbor, MI
48109, USA. 3Department of Internal Medicine, University of
Michigan Medical School, Ann Arbor, MI 48109, USA.
4Department of Microbiology and Immunology, University of
Michigan Medical School, Ann Arbor, MI 48109, USA.
5Departments of Biomedical Engineering and Mechanical
Engineering, University of Michigan Medical School, Ann
Arbor, MI 48109, USA. 6Department of Pathology and
Committee on Immunology, University of Chicago, Chicago,
IL 60637, USA. 7Institute for Advanced Biosciences, Keio
University, Yamagata, Japan. 8PRESTO, Japan Science and
Technology Agency, Kawaguchi, Saitama 332-0012, Japan.
*These authors contributed equally to this work. †Corresponding
author. Email: firstname.lastname@example.org (G.N.); yungikim77@
gmail.com (Y.-G.K.) ‡Present address: Division of Biochemistry,
Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku,
Tokyo 105-8512, Japan. §Present address: Department of Biomedical Sciences, Wide River Institute of Immunology, Seoul
National University College of Medicine, Seoul 03080, Korea.